Identification And Characterization Of A Class Ⅰ Newcastle Disease Virus Strain Isolated From Waterfowl In China

Newcastle disease, a notifiable disease which must to be reported to the World Organization for Animal Health (OIE), can cause severe economic losses in many countries. Newcastle disease virus (NDV) has historically been grouped into either genotypes or genetic lineages under one serotype (APMV-1). Recent analysis of genome sizes and sequences have revealed two distinct clades within APMV-1: ClassⅠand ClassⅡ. In recent years, ClassⅠNDVs were reported to be isolated from wild waterfowl and live bird markets in North America, Europe, and Hong Kong and so on. Its origin and role in the incidence and prevalence of ND in waterfowl have attracted many scholars.One ClassⅠNDV was isolated from healthy ducks in China in 2008 and characterized biologically and genotypically by National Reference Laboratory for Newcastle disease. The complete sequence of genome was established by sequencing cDNA fragments generated by high-fidelity reverse transcription-polymerase chain reaction (RT-PCR), and the antigenic homology among different genotype NDVs was compared through Cross-HI test. The results revealed the isolate was classified into genotype 3 in ClassⅠ, and the entire genomic sequence consists of 15198 nucleotides. Comparison of the complete genomic sequences showed that the isolate is only distantly related to the members of ClassⅡNDVs. Cross-HI tests of four NDV strains showed that HI homology among them is relatively low.1. Isolation, identification and partial biological characterization of one ClassⅠNewcastle Disease Virus.The virus was isolated from healthy ducks and identified by HI test using NDV and Avian Influenza virus (H5 and H9) positive serum and high-fidelity reverse transcription-polymerase chain reaction (RT-PCR). At the same time, it was purified by plaque-purification test, and characterized biologically. Based on its character of no lethal for 9-11 days SPF chicken embryos, ICPI (0.29), and the sequence of fusion protein cleavage site (112E-Q-Q-E-R-L117), the isolate was predicted to be lentogenic. Phylogenetic analysis of 57 Class I NDV isolates revealed that the isolate was similar to the viruses isolated from live bird markets in Honk Kong in recent years, and all of them were classified into the genotype 3 in ClassⅠ. It is named ND08-004.2. Genome and genetic characterization analyses of Newcastle Disease Virus ND08-004 strainComparing the isolate with sequences of NDV genome released in GenBank, 8 pairs of primers were designed and utilized to acquire overlapping viral cDNA fragments of ND08-004 strain by RT-PCR. The sequence of the 3′- and 5′terminal ends of viral RNA genome were determined by sequencing cDNA fragments generated by rapid amplification of cDNA ends (RACE). The entire genomic sequence consisted of 15198 nucleotides due to a 12 nucleotide insert in the coding region of the phosphoprotein gene between the corresponding position 495 and 496nt.3. Antigenic homology comparison of different Newcastle Disease Virus through Cross HI testSPF chickens were immunized with inactivated oil-emulsion vaccines of Ningxia strain,ND08-004, F48E8 and Lasota strains, and four kinds of hyperimmune serum were obtained 30 days later. Cross-HI was conducted through these Viruses and their correspondent hyperimmune serum. The results showed that HI homology between ND08-004 and LaSota, F48E8 and Ningxia strains is 71.42%, 66.82% and 57.14% respectively. Based on the results relatively diversities are found between ND08-004 strain and the ClassⅡNDVs concerned.